The invention relates to devices for separating and resuspending magnetic or magnetizable particles from liquids by means of a magnetic field produced by one or more permanent magnets.
The invention further relates to methods for separating magnetic or magnetizable particles from liquids and to the mixing and resuspending of magnetic or magnetizable particles in liquids by means of a magnetic field produced by one or more permanent magnets. The devices and methods can be used, for example, for applications in drug development, biochemistry, molecular genetics, microbiology, medical diagnostics and forensic medicine.
Methods that are based on magnetic separation using specifically binding, magnetically attractable particles or that enable the mixing of magnetic or magnetizable particles in solution are increasingly gaining in significance in the field of sample preparation for diagnostic or analytic examinations or in the field of performing diagnostic or analytic examinations. This is true, in particular, for automated processes since it is thereby possible to prepare or analyze a large number of samples within a short period of time and to dispense with labor-intensive centrifugation steps. This creates the conditions required for efficient, low-cost screening at a high sample throughput, which is extremely important for applications in molecular-genetic studies or in the field of medical diagnostics, for example, as it is practically impossible to manage or to pay for a purely manual handling of very large numbers of samples. Further important fields of application relate to pharmaceutical screening methods for identification of potential pharmaceutical active agents.
The basic principle of magnetic separation of substances from complex mixtures is based on the process of functionalizing magnetic particles (magnetizable or magnetically attractable particles) in a specific manner for the intended separation process, that is, they are provided, by chemical treatment, with specific binding properties for the target substances to be separated. The size of these magnetic particles is typically in the range of approx. 0.05 to 500 μm.
Magnetic particles that have specific binding properties for certain substances and can be used to remove these substances from complex mixtures are described, for example, in German published patent application DE 195 28 029 A1 and are commercially available (e.g. from chemagen Biopolymer-Technologie AG, DE-52499 Baesweiler, Germany).
In known separating methods the functionalized magnetic particles are added in a first step (“binding step”) to a mixture to be purified which contains the target substance(s) in a liquid promoting the binding of the target substance molecules to the magnetic particles (binding buffer). This causes a selective binding of the target substance(s) present in the mixture to the magnetic particles. Subsequently, these magnetic particles are immobilized (as a “pellet”) on a site of the interior wall of the reaction vessel by employing magnetic forces, that is, a magnetic field, for instance by means of a permanent magnet. Thereafter, the liquid supernatant is separated and discarded, for example by suction or decanting. Since the magnetic particles are immobilized in the manner described, it is largely prevented that these particles are removed along with the supernatant.
Subsequently, the immobilized magnetic particles are again resuspended. For this purpose an eluting liquid or eluting buffer is used that is suitable for breaking the bond between the target substance(s) and the magnetic particles, so that the target substance molecules can be released from the magnetic particles and removed along with the elution liquid while the magnetic particles are immobilized by the action of the magnetic field. One or more washing steps may be carried out prior to the elution step.
If appropriately functionalized, the magnetic particles can also be utilized directly for diagnostic or analytical examinations. In this case, functionalization enables the specific binding, for example, of pathogenic substances. However, in order to be able to make a statement that is as definite as possible, e.g. with regard to a pathogenic substance, suitable solutions must be freed from all impurities. To this end, the particles, to which the analytes adhere, must be mixed (washed) as efficiently as possible. The present invention facilitates such a process, particularly if there are large numbers of samples to be treated simultaneously or if one has to work with small volumes (384 or 1536 formats).
Devices of various types have been described for carrying out separation processes by means of magnetic particles. German utility model DE 296 14 623 U1 discloses a magnetic separator provided with movable permanent magnets. As an alternative it is proposed to move the reaction vessel containing the magnetic particles, by mechanical drive means, relative to a fixedly mounted permanent magnet. The device described in DE 296 14 623 U1 does not have magnetizable bars that are immersed in the sample liquid; rather, the permanent magnets are positioned next to the individual reaction vessels.
The device described in German published patent application DE 100 63 984 A1, which is provided with a magnet holder and a movable reaction vessel holder, also works according to a similar principle, it being possible to position the magnets laterally at the reaction vessels.
By using the above-mentioned devices it is possible to immobilize or accumulate the magnetic particles on the interior wall or on the bottom of a reaction vessel as a “pellet”. These devices are, however, not suitable for removing the magnetic particles from a reaction vessel. As a consequence it is necessary to exhaust the liquid from each individual reaction vessel by suction in order to separate the liquid from the magnetic particles. This is a disadvantage as it entails high material consumption (disposable pipette tips). Furthermore, it is unavoidable that individual magnetic particles are also sucked off, thus leading to a high error rate. Other errors can be caused by liquids dripping down, leading to cross-contamination.
German Patent DE 100 57 396 C1 proposes a magnetic separator provided with a plurality of rotatable bars that can be magnetized by an electromagnetic excitation coil. By immersing the bar in the liquid containing magnetic particles and withdrawing the bar in the magnetized state, the magnetic particles can be removed from the liquid and, if required, transferred to another reaction vessel where they can be re-released into a liquid, e.g. a wash or elution liquid, by deactivating the excitation coil.
A disadvantage of this device is that the excitation coil requires a relatively large space, which results in limitations of design and construction.
In addition, the positioning as well as the number of the bars is dependent on the geometry of the electromagnet, which may lead to limitations in the processing of samples. However, the geometry of the electromagnet cannot be altered arbitrarily as this would mean that inhomogeneity of the magnetic field would have to be accepted.
The known devices are, above all, not suitable for treating larger numbers of samples, as is required for high-throughput applications (e.g. microtiter plates with 364 or 1536 wells). The effort and expenditure in terms of construction would be immense, and, in addition, one would have to accept a significantly higher susceptibility to malfunction of the mechanical equipment employed.
Furthermore, the known devices are disadvantageous since they are suitable only for individual sample vessels or only for a certain, unalterable, pre-determined arrangement of sample vessels, e.g. in the form of a 96-well microtiter plate. However, for practical purposes it is desirable that such a magnetic separator device be suitable for, or can be converted for different types of sample vessels or for different arrangements of sample vessels (e.g. microtiter plates with 96, 364 or 1536 wells).